1. Field of the Invention
The present invention relates to the characterization of the binding site involved in binding between a binding protein and a binding partner.
2. Background Art
Biochemical Binding, Generally
Many biological processes are mediated by noncovalent binding interactions between a protein and another molecule, its binding partner. The identification of the structural features of the two binding molecules which immediately contribute to those interactions would be useful in designing drugs which alter these processes.
The molecules which preferentially bind each other may be referred to as members of a "specific binding pair". Such pairs include an antibody and its antigen, a lectin and a carbohydrate which it binds, an enzyme and its substrate, and a hormone and its cellular receptor. In some texts, the terms "receptor" and "ligand" are used to identify a pair of binding molecules. Usually, the term "receptor" is assigned to a member of a specific binding pair which is of a class of molecules known for its binding activity, e.g., antibodies. The term "receptor" is also preferentially conferred on the member of the pair which is larger in size, e.g., on avidin in the case of the avidin-biotin pair. However, the identification of receptor and ligand is ultimately arbitrary, and the term "ligand" may be used to refer to a molecule which others would call a "receptor". The term "anti-ligand" is sometimes used in place of "receptor".
While binding interactions may occur between any pair of molecules, e.g., two strands of DNA, the present specification is primarily concerned with interactions in which at least one of the molecules is a protein. Hence, it is convenient to speak of a "binding protein" and its "binding partner". The term "protein" is used herein in a broad sense which includes, mutatis mutandis, polypeptides and oligopeptides, and derivatives thereof, such as glycoproteins, lipoproteins, and phosphoproteins. The essential requirement is that the "binding protein" feature one or more peptide (--NHCO--) bonds, as the amide hydrogen of the peptide bond (as well as in the side chains of certain amino acids) has certain properties which lends itself to analysis by proton exchange.
A "binding site" is a point of contact between a binding surface ("paratope") of the binding protein and a complementary surface ("epitope") of the binding partner. (When the binding partner is a protein, the designation of "paratope" and "epitope" is essentially arbitrary. However, in the case of antibody-antigen interactions, it is conventional to refer to the antigen binding site of the antibody as the "paratope" and the target site on the antigen as the "epitope".) A specific binding pair may have more than one binding site, and the term "pair" is used loosely, as the binding protein may bind two or more binding partners (as in the case of a divalent antibody). Moreover, other molecules, e.g., allosteric effectors, may alter the conformation of a member of the "pair" and thereby modulate the binding. The term "pair" is intended to encompass these more complex interactions.
Limitations of Current Methods of Characterizing Protein Binding Sites
Considerable experimental work and time are required to precisely characterize a binding site. In general, the techniques which are the easiest to use and which give the quickest answers, result in an inexact and only approximate idea of the nature of the critical structural features. Techniques in this category include the study of proteolytically generated fragments of the protein which retain binding function; recombinant DNA techniques, in which proteins are constructed with altered amino acid sequence (site directed mutagenesis); epitope scanning peptide studies (construction of a large number of small peptides representing subregions of the intact protein followed by study of the ability of the peptides to inhibit binding of the ligand to receptor); covalent crosslinking of the protein to its binding partner in the area of the binding site, followed by fragmentation of the protein and identification of crosslinked fragments; and affinity labeling of regions of the receptor which are located near the ligand binding site of the receptor, followed by characterization of such "nearest neighbor" peptides. (Reviewed in 1, 2).
These techniques work best for the determination of the structure of binding subregions which are simple in nature, as when a single short contiguous stretch of polypeptide within a protein is responsible for most of the binding activity. However, for many protein-binding partner systems of current interest, the structures responsible for binding on both receptor and ligand or antibody are created by the complex interaction of multiple non-contiguous peptide sequences. The complexities of these interactions may confound conventional analytical techniques, as binding function is often lost as soon as one of the 3-dimensional conformations of the several contributing polypeptide sequences is directly or indirectly perturbed.
The most definitive techniques for the characterization of the structure of receptor binding sites have been NMR spectroscopy and X-ray crystallography. While these techniques can ideally provide a precise characterization of the relevant structural features, they have major limitations, including inordinate amounts of time required for study, inability to study large proteins, and, for X-ray analysis, the need for protein-binding partner crystals (Ref. 3).
Applicant's technology overcomes these limitations and allows the rapid identification of each of the specific polypeptides and amino acids within a protein which constitute its protein ligand binding site or antibody binding subregion in virtually any protein-ligand system or protein antigen-antibody system, regardless of the complexity of the binding sites present or the size of the proteins involved. This technology is superior in speed and resolution to currently employed biochemical techniques.
Tritium Exchange
When a protein in its native folded state is incubated in buffers containing tritiated water, tritium in the buffer reversibly exchanges with hydrogen present in the protein at acidic positions (for example, O--H, S--H, and N--H groups) with rates of exchange which are dependent on each exchangeable proton's chemical environment, temperature, and most importantly, its accessibility to the tritiated water in the buffer. (Refs. 4, 5) Accessibility is determined in turn by both the surface (solvent-exposed) disposition of the proton, and the degree to which it is hydrogen-bonded to other regions of the folded protein. Simply stated, acidic protons present on amino acid residues which are on the outside (buffer-exposed) surface of the protein and which are hydrogen-bonded to solvent water will exchange more rapidly with tritium in the buffer than will similar acidic protons which are buried and hydrogen-bonded within the folded protein.
Proton exchange reactions can be greatly accelerated by both acid and base-mediated catalysis, and the rate of exchange observed at any particular pH is the sum of both acid and base mediated mechanisms. For many acidic protons, a pH in the range of 2.7 results in an overall minimum rate of exchange (Ref. 6, pg.238, FIG. 3, refs. 7-11). While hydrogens in protein hydroxyl and amino groups exchange with tritium in buffer at millisecond rates, the exchange rate of one particular acidic proton, the peptide amide bond proton, is considerably slower, having a half life of exchange (when freely hydrogen bonded to solvent water) of approximately 0.5 seconds at 0.degree. C., pH 7, which is greatly slowed to a half life of exchange of 70 minutes at 0.degree. C. pH 2.7.
When peptide amide protons are buried within a folded protein, or are hydrogen bonded to other parts of the protein, exchange half lives with solvent protons are often considerably lengthened, at times being measured in hours to days. Proton exchange at peptide amides is a fully reversible reaction, and rates of on-exchange (solvent tritium replacing protein-bound hydrogen) are identical to rates of off-exchange (hydrogen replacing protein-bound tritium) if the state of a particular peptide amide within a protein, including its chemical environment and accessibility to solvent protons, remains identical during on-exchange and off-exchange conditions.
Tritium exchange techniques have been extensively used for the measurement of peptide amide exchange rates within an individual protein (reviewed in 4). The rates of exchange of other acidic protons (OH, NH, SH) are so rapid that they cannot be followed in these techniques and all subsequent discussion refers exclusively to peptide amide proton exchange. In these studies, purified proteins are on-exchanged by incubation in buffers containing tritiated water for varying periods of time, transferred to buffers free of tritium, and the rate of off-exchange of tritium determined. By analysis of the rates of tritium on- and off-exchange, estimates of the numbers of peptide amide protons in the protein whose exchange rates fall within particular exchange rate ranges can be made. These studies do not allow a determination of the identity (location within the protein's primary amino acid sequence) of the exchanging amide hydrogens measured.
Extensions of these techniques have been used to detect the presence within proteins of peptide amides which experience allosterically-induced changes in their local chemical environment and to study pathways of protein folding (5, 12-14). For these studies, tritium on-exchanged proteins are allowed to off-exchange after they have experienced either an allosteric change in shape, or have undergone time-dependent folding upon themselves, and the number of peptide amides which experience a change in their exchange rate subsequent to the allosteric/folding modifications determined. Changes in exchange rate indicate that alterations of the chemical environment of particular peptide amides have occurred which are relevant to proton exchange (solvent accessibility, hydrogen bonding etc.). Peptide amides which undergo an induced slowing in their exchange rate are referred to as "slowed amides" and if previously on-exchanged tritium is sufficiently slowed in its off-exchange from such amides there results a "functional tritium labeling" of these amides. From these measurements, inferences are made as to the structural nature of the shape changes which occurred within the isolated protein. Again, determination of the identity of the particular peptide amides experiencing changes in their environment is not possible with these techniques.
Four groups of investigators have described technical extensions (collectively referred to as medium resolution tritium exchange) which allow the locations of particular slowed, tritium labeled peptide amides within the primary sequence of small proteins to be localized to a particular proteolytic fragment, though not to a particular amino acid.
Rosa and Richards were the first to describe and utilize medium resolution tritium techniques in their studies of the folding of ribonuclease S protein fragments (15-17). However, the techniques described by Rosa and Richards were of marginal utility, primarily due to their failure to optimize certain critical experimental steps (reviewed in 6, pg 238, 244). No studies employing related techniques were published until the work of Englander and co-workers in which extensive modifications and optimizations of the Rosa and Richards technique were first described.
Englander's investigations utilizing tritium exchange have focused exclusively on the study of allosteric changes which take place in tetrameric hemoglobin (.alpha. subunit and .beta. subunit 16 kD in size each) upon deoxygenation (6,18-21). In the Englander procedure, native hemoglobin (milligram quantities) in the oxygenated state is on-exchanged in tritiated water of relatively low specific activity (2-100 mCi/ml). The hemoglobin is then deoxygenated (inducing allosteric change), transferred to tritium-free buffers by gel permeation column chromatography, and then allowed to out-exchange for 10-50 times the on-exchange time. On-exchanged tritium present on peptide amides which experience no change in exchange rate subsequent to the induced allosteric change in hemoglobin structure off-exchanges at rates identical to its on-exchange rates, and therefore is almost totally removed from the protein after the long off-exchange period. However, peptide amides which experience slowing of their exchange rate subsequent to the induced allosteric changes preferentially retain the tritium label during the period of off-exchange.
To localize (in terms of hemoglobin's primary sequence) the slowed amides bearing the residual tritium label, Englander then proteolytically fragments the off-exchanged hemoglobin with the protease pepsin, separates, isolates and identifies the various peptide fragments by reverse phase high pressure liquid chromatography (RP-HPLC), and determines which fragments bear the residual tritium label by scintillation counting. However, as the fragmentation of hemoglobin proceeds, each fragment's secondary and tertiary structure is lost and the unfolded peptide amides become freely accessible to H.sub.2 O in the buffer. At physiologic pH (&gt;6), any amide-bound tritium label would leave the unfolded fragments within seconds. Englander therefore performs the fragmentation and HPLC peptide isolation procedures under conditions which he believes minimize peptide amide proton exchange, including cold temperature (4.degree. C.) and use of phosphate buffers at pH 2.7 (reviewed in 6). This technique has been used successfully by Englander to coarsely identify and localize the peptide regions of hemoglobin .alpha. and .beta. chains which participate in deoxygenation-induced allosteric changes (18-21). The ability of the Englander technique to localize tritium labeled amides, while an important advance, remains low; at the best, Englander reports that his technique localizes amide tritium label to hemoglobin peptides 14 amino acids or greater in size, without the ability to further sublocalize the label.
In Englander's work, there is no appreciation that a suitably adapted tritium exchange technique might be used to identify the peptide amides which reside in the contacting surface of a protein receptor and its binding partner: his disclosures are concerned exclusively with the mapping of allosteric changes in hemoglobin. Furthermore, based on his optimization studies (6-11, 13), Englander teaches and warns that a pH of 2.7 must be employed in both the proteolysis and HPLC steps, necessitating the use of proteases which are functional at these pH's (acid proteases). Unfortunately, acid proteases are relatively nonspecific in their sites of proteolytic cleavage, leading to the production of a very large number of different peptide fragments and hence to considerable HPLC separation difficulties. The constraint of performing the HPLC separation step at pH 2.7 greatly limits the ability to optimize the chromatographic separation of multiple overlapping peptides by varying the pH at which HPLC is performed. Englander tried to work around these problems, for the localization of hemoglobin peptides experiencing allosteric changes, by taking advantage of the fact that some peptide bonds are somewhat more sensitive to pepsin than others. He therefore limits the duration of exposure of the protein to pepsin to reduce the number of fragments. Even then the fragments were "difficult to separate cleanly". They were also, of course, longer (on average), and therefore the resolution was lower. He also tried to simplify the patterns by first separating the alpha and beta chains of hemoglobin. However, there was a tradeoff: increased tritium loss during the alpha-beta separation and the removal of the solvent, preparatory to proteolysis. Englander concludes,
"At present the total analysis of the HX (hydrogen exchange) behavior of a given protein by these methods is an immense task. In a large sense, the best strategies for undertaking such a task remain to be formulated. Also, these efforts would benefit from further technical improvements, for example in HPLC separation capability and perhaps especially in the development of additional acid proteases with properties adapted to the needs of these experiments" (6).
Over the succeeding seven years since this observation was made, no advances have been disclosed which address these critical limitations of the medium resolution tritium exchange technique. It has been perceived that improvements to the HPLC separation step were problematic due to the constraint of working at pH 2.7. The current limited success with small proteins has made it pointless to attempt similar studies of larger proteins where the problems of inadequate HPLC peptide separation at pH 2.7, and imprecision in the ability to sublocalize labeled amides would be greatly compounded. Furthermore, most acid-reactive proteases are in general no more specific in their cleavage patterns than pepsin and efforts to improve the technology by employing other acid reactive proteases other than pepsin have not significantly improved the technique. Given these limitations of medium resolution tritium exchange art, no studies have been disclosed which utilize proteins with subunit size greater than 16 kilodaltons.
Allewell and co-workers have disclosed studies utilizing the Englander techniques to localize induced allosteric changes in the enzyme escherichia coli aspartate transcarbamylase (22,23). Burz, et al. (22) is a brief disclosure in which the isolated R2 subunit of this enzyme is on-exchanged in tritiated buffer of specific activity 100 mCi/ml, allosteric change induced by the addition of ATP, and then the conformationally altered subunit off-exchanged. The enzyme R2 subunit was then proteolytically cleaved with pepsin and analyzed for the amount of label present in certain fragments. Analysis employed techniques which rigidly adhered to the recommendations of Englander, utilizing a single RP HPLC separation in a pH 2.8 buffer.
The authors note difficulty in separating the large number of peptides generated, even from this small protein subfragment, given the constraints of the Englander methodology. They comment that "the principal limitation of this method at present is the separation with columns now available". ATP binding to the enzyme was shown to alter the rate of exchange of hydrogens within several relatively large peptidic fragments of the R2 subunit. In a subsequent more complete disclosure (23), the Allewell group discloses studies of the allosteric changes induced in the R2 subunit by both ATP and CTP. They disclose on-exchange of the R2 subunit in tritiated water-containing buffer of specific activity 22-45 mCi/ml, addition of ATP or CTP followed by off exchange of the tritium in normal water-containing buffer. The analysis comprised digestion of the complex with pepsin, and separation of the peptide fragments by reverse phase HPLC in a pH 2.8 or pH 2.7 buffer, all of which rigidly adheres to the teachings of Englander. Peptides were identified by amino acid composition or by N-terminal analysis, and the radioactivity of each fragment was determined by scintillation counting. In both of these studies the localization of tritium label was limited to peptides which averaged 10-15 amino acids in size, without higher resolution being attempted.
Finally, Beasty, et al. (24) have disclosed studies employing tritium exchange techniques to study folding of the .alpha. subunit of E. Coli tryptophan synthetase. The authors employed tritiated water of specific activity 20 mCi/ml, and fragmented the tritium labeled enzyme protein with trypsin at a pH 5.5, conditions under which the protein and the large fragments generated retained sufficient folded structure as to protect amide hydrogens from off exchange during proteolysis and HPLC analysis. Under these conditions, the authors were able to produce only 3 protein fragments, the smallest being 70 amino acids in size. The authors made no further attempt to sublocalize the label by further digestion and/or HPLC analysis. Indeed, under the experimental conditions they employed (they performed all steps at 12.degree. C. instead of 4.degree. C., and performed proteolysis at pH 5.5 instead of pH in the range of 2-3), it would have been impossible to further sublocalize the labeled amides by tritium exchange, as label would have been immediately lost (off-exchanged) by the unfolding of subsequently generated proteolytic fragments at pH 5.5 if they were less than 10-30 amino acids in size.
In summary, the above disclosures are restricted to studies of medium resolution tritium exchange of: 1) The re-folding on itself of different parts of an individual protein (tryptophan synthetase .alpha. subunit) (24); 2) The re-folding onto itself of two fragments proteolytically generated from the same protein (ribonuclease-S) (15-17); 3) The changes in shape (allosteric change) which an individual protein (hemoglobin) underwent subsequent to removal of oxygen (hemoglobin) (4-6,12-14,18-21); and 4) The allosteric changes in a protein after the addition of known allosteric change inducers (aspartate transcarbamylase) (22,23).
Because tritium exchange art was limited in its ability to study large proteins, none of these or other investigators disclosed or proposed that tritium exchange techniques could be adapted to effectively study contact surfaces between two different, large proteins (subunits &gt;16 kD in size) or that peptide amides functionally labeled with tritium in large protein-binding partner interactions could effectively be localized precisely at the amino acid sequence level.
Fromageot, et al., U.S. Pat. No. 3,828,102 (25) discloses using hydrogen exchange to tritium label a protein and its binding partner. The protein-binding partner complex is formed before allowing on-exchange to occur and thus the binding site is not selectively labeled. In the present invention the protein is on-exchanged before its interaction with binding partner and subsequent off-exchange, and thus, the peptide amides which reside in the interactions surface specifically retain label while other sites do not.
Benson, U.S. Pat. Nos. 3,560,158 and 3,623,840 (26) disclose using hydrogen exchange to tritiate compounds for analytical purposes. These references differ from the invention by not providing any mechanism for distinguishing between any potential binding site and the rest of the molecule.
NMR-Deuterium Techniques to Study Protein-Binding Partner Interactions: Fesik, et al (27) discloses measuring by NMR the hydrogen (deuterium) exchange of a peptide before and after it is bound to a protein. From this data, the interactions of various hydrogens in the peptide with the binding site of the protein are analyzed.
Patterson, et al. (28) and Mayne, et al. (29) disclose NMR mapping of an antibody binding site on a protein (cytochrome-C) using deuterium exchange. This relatively small protein, with a solved NMR structure, is first complexed to anti-cytochrome-C monoclonal antibody, and the preformed complex then incubated in deuterated water-containing buffers and NMR spectra obtained at several time intervals. The NMR spectra of the antigen-antibody complex is examined for the presence of peptide amides which experience slowed hydrogen exchange with solvent deuterium as compared to their rate of exchange in uncomplexed native cytochrome-C. Benjamin, et al. (30) employ an identical NMR-deuterium technique to study the interaction of hen egg lysosozyme (HEL) with HEL-specific monoclonal antibodies. While both this NMR-deuterium technique, and medium resolution tritium exchange rely on the phenomenon of proton exchange at peptide amides, they utilize radically different methodologies to measure and localize the exchanging amides. Furthermore, study of proteins by the NMR technique is not possible unless the protein is small (less than 30 kD), large amounts of the protein are available for the study, and computationally intensive resonance assignment work is completed.
Recently, others (45-50) have disclosed techniques in which exchange-deuterated proteins are incubated with binding partner, off-exchanged, the complex fragmented with pepsin, and deuterium-bearing peptides identified by single stage Fab or electrospray MS. In these studies, no attempt has been made to sublocalize peptide-bound deuterium within pepsin-generated peptides.